The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef works via host cell signaling proteins including Hck and other Src-family kinases (SFKs), suggesting that Nef-SFK complexes are present in all HIV-infected cell types. We have recently identified small molecules that selectively target Nef-SFK complexes as well as the Nef protein itself. These compounds also potently inhibit HIV-1 replication in a Nef-dependent manner, indicating that they interfere with Nef function in an HIV-relevant manner. The major goals of this proposal, therefore, are to solve X-ray crystal structures of Nef in complexes with full-length SFKs for the first time, and to map inhibitor binding sites within these complexes. These structures are critical to a full understanding of the mechanisms of compound action, which will be explored in complementary functional assays. These goals will be pursued with the following Specific Aims: Aim 1. Determine the X-ray crystal structures of HIV-1 Nef-SFK complexes in the presence of selective inhibitors. Our previous work suggests that Nef binding allosterically induces a unique conformation at the Hck active site, providing a specific target for small molecules. To test the impact of Nef binding on the conformation of the Hck kinase domain, we will solve the crystal structures of Nef-Hck protein complexes in the presence of 4- amino-substituted diphenylfuranopyrimidine (DFP) kinase inhibitors, which we recently reported as selective inhibitors of both Nef-induced Hck activity and Nef-dependent HIV-1 replication. Aim 2. Determine the X-ray crystal structures of HIV-1 Nef with newly identified antagonists of Nef function. Coupling of HIV-1 Nef to Hck activation enabled a large-scale fully automated screen and discovery of first-in-class Nef antagonists with antiretroviral activity. Preliminary biophysical studies demonstrate the direct nanomolar binding of these compounds to Nef. Here we will identify the binding sites for these compounds by co-crystallizing them with HIV-1 Nef, and validate the structures through biochemical studies with binding site mutants. Aim 3. Test structural models of Nef-directed antiretroviral compounds in HIV replication. Based on existing data, as well as new structural information to be obtained in the first two Aims, we will create Nef mutants that are predicted to be resistant to the actions of our compounds. These mutations will be engineered into the HIV-1 backbone, and their impact on HIV-1 sensitivity to each compound will be assessed in replication assays. In addition, we will generate and test new analogs of each lead compound based on structural data to improve solubility, potency and pharmacological properties. These studies will provide important mechanistic evidence that the compounds antagonize Nef functions relevant to HIV-1 in a manner predicted by the structural biology.